Tri kit

RNA extraction was performed using a TRI kit (Sigma), according to the kit‘s instructions, using the bone marrow mononuclear cells (MNCs) of both the patients and control groups. Before running qPCR, the same RNA concentration was established for all samples.
For the gene expression study, quantitative PCR was performed in triplicate for both the patient and control groups using Realplex Sequence Detection System (Eppendorf, Hauppauge, NY, USA) Step PlusOne thermal cycler (Thermo Fisher Scientific, Waltham, MA) with the following setting: 95 °C for 10 min, followed by 40 cycles of 95 °C for 15 sec, and then 60 °C for 1 min. The comparative CT (2-ΔΔCt) method was used to study the relative gene expressions in both groups. We used NF-κB, and HTRP (housekeeping gene, used as internal control) primers from Invitrogen (Grand Island, NY, USA) (Table 3).

For PCR analysis of human bone marrow mononuclear cells (MNCs), the total RNA was extracted using a TRI kit (Sigma) according to the manufacturer's instructions. Complementary DNA (cDNA) was synthesized with the same concentration of RNA in all samples and subjected to qPCR using Realplex Sequence Detection System (Eppendorf, Hauppauge, NY, USA) StepPlusOne thermal cycler (Thermo Fisher Scientific, Waltham, MA) with settings: 95°C for 10 min, followed by 40 cycles of 95°C for 15 s, and 60°C for 1 min. The primers for CD34, TIM-3, CLL-1, BMI-1, and HTRP primer, house-keeping gene (used as internal control) (Table 1) were obtained from Invitrogen (Grand Island, NY, USA).
All the reactions were performed in triplicate with 20 μl Taqman Universal PCR Master Mix (KAPA Biosystem, Wilmington, MA, USA) containing 1.0 μl cDNA. The relative gene expressions in controls and patients were determined by using the comparative CT (2-ΔΔCt) method.